Co-pyrolysis of chicken manure with tree bark for reduced biochar toxicity and enhanced plant growth in Arabidopsis thaliana

Co-pyrolysis of chicken manure with tree bark was investigated to mitigate salinity and potentially toxic element (PTE) concentrations of chicken manure-derived biochar. The effect of tree bark addition (0, 25, 50, 75 and 100 wt%) on the biochar composition, surface functional groups, PTEs and polycyclic aromatic hydrocarbons (PAH) concentration in the biochar was evaluated. Biochar-induced toxicity was assessed using an in-house plant growth assay with Arabidopsis thaliana. This study shows that PTE concentrations can be controlled through co-pyrolysis. More than 50 wt% of tree bark must be added to chicken manure to reduce the concentrations below the European Biochar Certificate-AGRO (EBC-AGRO) threshold. However, the amount of PAH does not show a trend with tree bark addition. Furthermore, co-pyrolysis biochar promotes plant growth at different application concentrations, whereas pure application of 100 wt% tree bark or chicken manure biochar results in decreased growth compared to the reference. In addition, increased plant stress was observed for 100 wt% chicken manure biochar. These data indicate that co-pyrolysis of chicken manure and tree bark produces EBC-AGRO-compliant biochar with the potential to stimulate plant growth. Further studies need to assess the effect of these biochars in long-term growth experiments.


The analysis of the feedstock blends and the resulting biochars
The thermal behaviour of the feedstock blends (5-10 mg) was analysed by thermogravimetric analysis (TGA, Q500 TA instruments) according to the same procedure described in our previous publication 16 .In short, the sample was subjected to the following temperature program: (1) temperature ramp from 25 to 600 °C in N 2 (20 °C/min), (2) gas flow was switched from N 2 to O 2 , (3) isothermal step at 600 °C in O 2 and (4) temperature ramp from 600 to 900 °C in O 2 .
The pH and electrical conductivity (EC) (mS/cm) were measured in triplicate after a 24-h extraction in Milli-Q water (solid:liquid ratio 1:10) 16 .The macronutrient (P, K, Mg and Ca) and PTE (Pb, Cd, Cu, Ni, Hg, Zn, Cr and As) concentrations of the feedstock blends and biochars were analysed through ICP-OES (Perkin Elmer Optima 8300) after a microwave-assisted two-step digestion (Milestone Ethos) with concentrated nitric acid (HNO 3,conc ; 69 wt% p.a.; Suprapur; Merck) and hydrogen peroxide (H 2 O 2 ; 30 wt% p.a.; Supelco, Merck).In the first step, 7.0 mL HNO 3,conc and 3.0 mL H 2 O 2 were added to the sample (100 mg) in Teflon vessels.Secondly, 3.0 mL HNO 3,conc and 2.0 mL H 2 O 2 were added to the vessels.The microwave digestion was temperature controlled (220 °C, 20 °C/min with a holding time of 15 min).The digested samples were filtered and diluted with Table 1.The coding convention of the TB-CM feedstock blends and corresponding biochar used in this study.

Plant growth assay and biochar-induced stress assessment
Arabidopsis thaliana Col-0 wild-type seeds (Nottingham Arabidopsis Stock Centre (NASC)) were surface sterilised with 70 vol% ethanol and rinsed several times using sterile dH 2 O before being stored for 2 nights at 4 °C to allow imbibition and synchronise germination.Subsequently, seeds were sown in 96-well plates (Greiner Bio-One) with ¼ Murashige and Skoog (MS) medium (referred to as reference growth medium) and exposed to the different biochar blends BC-100-0, BC-75-25, BC-50-50, BC-25-75 and BC-0-100 at the following biochar concentrations: 0.00 wt%, 0.05 wt%, 0.10 wt%, 0.25 wt% and 0.50 wt% (one 96-well plate per condition).Seedlings were grown with a photoperiod of 12 h, a day/night temperature of 22/18 °C and a relative humidity of 65%.Photosynthetic active radiation (PAR) was approximately 170 μmol m 2 s −1 at the plant level and simulated using the GreenPower LED modules (Phillips) consisting of blue-, red-and far-red led modules.Four seedlings per sample and 8 biological replicates per condition were snap-frozen with liquid nitrogen seven days after sowing and stored at -80 °C for flow cytometry (FCM) analyses.Growth parameters like fresh weight and root length were determined of five seedlings per condition ten days after sowing.An in-house screening platform (plant growth assay) based on FCM analysis with the CyStain PI Absolute kit (Sysmex-Partec) was used to evaluate biochar-induced stress or growth promotion.Seedlings were chopped in 250 µL of nuclei extraction buffer and incubated for 1 min.Afterwards, the extract was filtered with a 50 µm CellTrics®.Subsequently staining solution consisting of 1 mL staining buffer, 6 µL PI and 3 µL RNAse A per sample was added to stain the DNA.After at least 1 h of incubation at 4 °C, samples were measured using a CyFlow Cube 8 Flow Cytometer (Sysmex-Partec).A 488 nm laser was used to excite the nuclei, and the forward scatter and PI fluorescence intensity (FL-2 channel, 580/30 nm) were measured.Data analysis was performed with the FCS Express 4 software (De Novo Software; https:// denov osoft ware.com/ full-access/ downl oad-landi ng/), and endoreplication indices for plant growth (EI growth ) and phytotoxicity (EI defence ) were calculated according to Cuypers et al. 29 .
To determine the leaching and adsorption of elements by biochar, biochars (BC-100-0, BC-75-25, BC-50-50, BC-25-75 and BC-0-100) were added to the medium at the same concentration levels as used for the plant growth experiments (0.00 wt%, 0.05 wt%, 0.10 wt%, 0.25 wt% and 0.50 wt%).Afterwards, the medium was stored under similar conditions as the plant growth experiments for ten days.Subsequently, the medium was filtered to remove the biochar, acidified with HNO 3,conc and ICP-OES was used to determine the dissolved K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ , Cu 2+ and total P.

Statistics
Statistical analyses were executed with R studio version 4.2.2 (https:// posit.co/ downl oad/ rstud io-deskt op/) 30 .Outliers were identified with the Grubbs test and deleted before further analysis.Normality and homoscedasticity of the data were checked with the Shapiro-Wilk test and the Barlett test, respectively.If normality was not fulfilled, the data's square root, inverse, exponential and logarithmic transformations were tested.If the data (or transformed data) met the assumptions, one-way ANOVA was used to identify significant differences between the different concentrations used within the same biochar type.When the data did not meet the assumptions, a nonparametric Kruskal-Wallis with post hoc Wilcoxon rank-sum exact test was executed.Furthermore, Pearson correlation coefficients were calculated after a visual inspection of normality with a qq-plot.

Ethics approval
The plant experiments were in accordance with relevant institutional, national, and international guidelines and legislations.Additionally, experiments do not violate the IUCN policy statement on Research Involving Species at Risk of Extinction and Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Biochar yield and characteristics
Figure 1 shows the biochar yield and ash content obtained after the pyrolysis experiment.The biochar yield varied between 40 (BC-100-0) and 45 wt% (BC-0-100), and the ash content varied between 4.7 (BC-100-0) and 53.9 wt% (BC-0-100).The relatively high biochar yield of BC-100-0 compared to woody and agricultural biochars in general (20-30 wt% biochar yield at a pyrolysis temperature of 450 °C) resulted from the high fixed carbon content of BM-100-0 (40 wt% on a dry and ash-free basis) (Table S1).In contrast, the biochar yield of BC-0-100 was more related to its high ash content (Fig. 1) 31 .The biochar yield of BC-75-25, BC-50-50 and BC-25-75 fluctuated between 42 and 45 wt% but within the pyrolysis experimental error (as a standard deviation: 4 wt%) 23 .When looking more closely at the volatile matter (57-62 wt% dm) (Table S1) and the thermogram (Fig. S1), there seems to be no large difference between the different feedstock blends around the pyrolysis temperature used (450 °C).Therefore, a similar biochar yield can be expected for the corresponding biochar.Although the biochar yield of the co-pyrolysis biochars was in a similar range, a difference in composition and biochar characteristics can be expected because of the increasing mineral fraction with CM addition.
Table 2 shows the co-pyrolysis biochars' elemental composition (TC-HNO), TC/N ratio, ash content, macronutrients (P, K, Ca and Mg), pH and EC.Total carbon (TC) content ranged from 37 wt% (BC-0-100) to 78 wt% (BC-100-0) and was negatively related to CM addition.This was substantiated by the increasing ash content with CM addition (Fig. 1) and also visible in the macronutrient concentration; CM addition caused significantly more P, K, Ca and Mg in the biochars, which increases their fertilisation potential.Similar trends were already observed for biochar produced through co-pyrolysis of pig manure and lignin 32 .The biochars' P, K, Ca and Mg concentrations varied respectively between 0.4-28.3mg/g, 2.3-34.8mg/g, 6-182 mg/g and 1.67-14 mg/g.The Ca concentration in the produced biochars reached a higher concentration than the other macronutrients.This suggests that the biochars can potentially be used as liming agents 33 .Biochar samples BC-100-0 and BC-75-25 demonstrated a high C/N ratio (> 35), indicating the potential to stimulate N mineralisation through microbial activity.In contrast, BC-25-75 and BC-0-100 had a low C/N ratio (< 20), favouring their use as a fertiliser.BC-50-50 showed an intermediate C/N ratio (28.2) which is believed to cause an optimum equilibrium state between N mineralisation and immobilisation 34 .The biochars showed an alkaline nature with a pH varying between 7.96 and 10.2.There seemed to be a stabilising effect around a pH of 10 for BC-25-75 and BC-0-100.This could be due to the acid-buffering effect of biochar induced by inorganic carbon (carbonate).Table S1 indeed shows that BM-25-75 and BM-0-100 have an increased carbonate concentration (4 wt% dm) compared to the other feedstock blends (< 1 wt% dm) based on the TGA thermogram.Our previous study highlighted the importance of the Ca and carbonate concentrations for the pH and acid-buffering capacity, respectively 23 .The addition of CM also caused an increase in the EC between 0.3 (BC-100-0) and 8.57 mS/cm (BC-0-100), which means the biochar has the potential to release more macronutrients into its environment.This was evaluated by incubating the biochars for 10 days in the reference growth medium at different concentrations (0.05, 010, 0.25 and 0.50 wt%).The concentration of the different leached nutrients (Na + , K + , Mg 2+ , Ca 2+ and total P) in the supernatant was analysed (Figs.S3-S5).The results indicated a high leaching of Na + and K + into the reference growth medium, especially for BC-25-75 and BC-0-100 (Fig. S3).For Na + , the concentration even increased by approximately one order of  magnitude at a biochar concentration of 0.50 wt%.The Mg 2+ concentration in the supernatant increased with an increasing biochar concentration.The Mg 2+ concentration in the growth medium was also controlled by the biochar's total Mg content, which increased with the increasing CM addition in the biochar's feedstock blend.
In contrast, the leaching of Ca 2+ showed a different trend; the biochar with the highest Ca content resulted in a low concentration in the growth medium and was generally higher for the biochars with the lowest Ca content.Furthermore, a decreasing and complete depletion of P in the growth medium was found with an increased biochar concentration of BC-100-0.This depletion of P in soils was also found in previous research 35 .This shows that the leaching of elements from biochars depends on different factors and is very complex.

The co-pyrolysis biochar surface functionalities
Figure 2 shows the FT-IR spectra of the co-pyrolysis biochars and their corresponding starting materials between 600 and 4000 cm −1 .The FT-IR spectrogram of the corresponding feedstock blends can be found in Figure S2.
The first broad peak in the grey region (approx.3000-3600 cm −1 ) corresponds to -OH stretching from different O-rich functional groups (-COOH, -OH, …).This peak was more present for the feedstock blends than the biochars because of the partial thermal degradation of O-rich functionalities.The two peaks at 2924 and 2851 are respectively associated with the asymmetric and symmetric stretching of alkyl (-CH x ) groups and are also found in all feedstock blends (Fig. S2).After pyrolysis, these peaks were inexistent and were substituted by a broader peak around 2887 cm −1 (a), representing alkylated surface groups.The peaks between 1700 and 1800 cm −1 (b) were small or existed as a shoulder peak in the spectra and can be assigned to the vibration of C=O 36 .The peak in the 1600 cm −1 region (c) is associated with C=C structures but can also correspond to the N-H stretching of amide groups 37,38 .This might be a plausible explanation as the biochars showed an increasing N content with CM addition 37,38 .However, this peak became a shoulder peak with CM addition, which contradicts that the peak originated from N species.Peaks around this region can also be attributed to -OH in-plane bending, C=O, and other oxygenated functionalities 39 .This could be substantiated because of the decreasing O content in the biochar with the increasing CM addition (Table 2).Interestingly the peaks around 1428 (d) and 872 (g) cm −1 , associated with carbonate (CO 3 2-), existed in the feedstock blends BM-75-25, BM-50-50, BM-25-75 and BM-0-100 and their corresponding biochars.The relative intensity of the peak at 872 cm −1 became more intense with the addition of CM, which further confirms that this peak can be attributed to CO 3 2− (Table S1) 40 .Besides CO 3 2− , the peaks 1428 (d) and 1034 (f) cm −1 can also be attributed to, respectively, C=C stretching and C-O-C asymmetric stretch 31 .For BC-100-0, these peaks did not exist, and only a broad band at 1165 (e) cm -1 was found that can be attributed to C-O stretching, as was also observed for tree bark-based biochar 41 .

Implications of PTEs during and after co-pyrolysis
As was stated by earlier studies, a major drawback of manure and sludge pyrolysis is the accumulation of PTEs in the biochar 22,25,42 .Some of these elements (Zn and Cu) are essential micronutrients for plants but become highly toxic at elevated concentrations.Other PTEs, such as Cr, are not beneficial for plants and induce toxicity.Table 3 shows the different biochars' Zn, Cu and Cr concentrations and their retention from feedstock after pyrolysis into biochars.The Zn, Cu and Cr concentrations varied respectively between 44.7-635 mg/kg, 66-184 mg/kg and 17.4-70 mg/kg.While the Zn retention in all biochars after pyrolysis was nearing 100 wt% (94 ± 6%), its Cu retention was lower (70 ± 8 wt%), which suggests that part of the Cu is transferred to the gas phase during pyrolysis.In contrast, the Cr retention reached up to 273 wt% (BC-100-0), which can be explained by Cr contamination from the stainless steel (type 316) reactor.The biochars BC-75-25, BC-50-50 and BC-25-75 showed a Cr retention between 102 and 129 wt% (113 ± 22 wt%), not significantly different from 100 wt% (p = 0.11).On the other hand, BC-0-100 showed a partial transfer of Cr to the gas phase through its low retention in the biochar (43 wt%).Only the biochars BC-25-75 and BC-0-100 showed a Zn and Cu concentration above the EBC-AGRO threshold (400 (Zn) and 100 (Cu) mg/kg).This means that 50 wt% of TB must be added to produce biochar that complies with this threshold.However, these PTEs need to be bioavailable to induce toxic effects in plants.In the case of the biochars used in this study, there was no additional leaching of Zn 2+ into the reference growth medium after 10 days of biochar incubation, regardless of the biochar concentration and biochar type (Fig. S6).There was even a decreasing trend with increasing biochar concentrations, evidencing some uptake of this element by the biochar.The reduction in the bioavailability of Zn by biochar was already evidenced 43 .The Cu and Cr concentrations in all (biochar-amended) growth media was not reported because both elements were not detected (Cu < 0.25 mg/L; Cr < 0.05 mg/L).This means that although the Zn 2+ and Cu 2+ concentrations in the biochars BC-25-75 and BC-0-100 exceeded the EBC-AGRO threshold, these biochars are not a source of PTEs in the current experiment.However, the long-term leaching effects of PTEs from biochar should be further assessed under various weathering conditions.
The formation and occurrence of PAHs during biomass pyrolysis still need to be fully understood.However, a previous study evidenced that the formation of some PAH constituents is affected by the interaction between cellulose, hemicellulose and lignin 45 .As the ratio of these compounds changed for the different feedstock blends, Table 3.The Zn, Cu and Cr concentrations and retention of the different biochars.The standard deviation of the Zn and Cu retention is calculated based on the error propagation theorem.The bold numbers exceed the EBC-agro limits.Other potentially toxic elements (Ni: < 10 mg/kg dm; Pb: < 10 mg/kg dm.Cd: < 1 mg/kg dm, Hg: < 1 mg/kg dm and As: < 10 mg/kg dm) were below the detection limit.n.d.: not detected.The values represent the average (standard deviation).www.nature.com/scientificreports/another interaction can be expected, potentially leading to an increased ∑16 EPA PAHs concentration.However, in the current pilot-scale set-up, the ∑16 EPA PAHs concentration in the biochar is also partly dependent on their condensation during biochar collection, as was proposed in a previous study 23 .
When combining the biochar toxicity data (PTEs, ∑16 EPA PAHs and ∑16 BAP-equivalent concentrations), up to 50 wt% CM can be added to the TB before pyrolysis to produce EBC-compliant biochar.In the case of BC-100-0, the high ∑16 EPA PAHs concentration resulted in non-compliance.Because of the complexity of PAH formation, this toxicity factor should continuously be assessed for different feedstock blends and feedstock ratios.

Plant growth and toxicity assessment
An in-house plant growth assay was used with A. thaliana as a model plant to assess biochar-induced phytotoxicity or its growth-promoting effects.Figure 4 shows the biometric parameters, i.e. plant fresh weight (Fig. 4a) and root length (Fig. 4b), as well as the relative EI growth (Fig. 4c) as an indicator for plant growth promotion and the relative EI defence (Fig. 4d) as an indicator for plant stress 29 .All biochars significantly affected the fresh weight (Fig. 4a) and root length (Fig. 4b) of A. thaliana seedlings compared to seedlings grown in the reference growth medium.However, this depends on the biochar blend used and the concentration applied.For each biochar, there is an optimal biochar concentration.In general, pure biochars, i.e.BC-100-0 and BC-0-100, had no positive effect on the fresh weight (except at 0.10 wt% application) and even significantly decreased plant fresh weight when 0.50 wt% BC-0-100 was added as compared to seedlings grown in the reference growth medium.A similar observation was seen for the biochar combinations with the higher CM addition (BC-50-50 and BC-25-75).On the other hand, the fresh weight of seedlings exposed to BC-75-25 increased compared to the seedlings grown in the reference growth medium, independent of the biochar concentration applied.Furthermore, the root length (Fig. 4b) of seedlings treated with BC-75-25 and BC-50-50 significantly increased for all biochar concentrations, except when 0.05 wt% was added.The 0.05 wt% concentration of BC-25-75 and BC-0-100 did increase the root length significantly.However, a decreasing trend in root length, down to the root length of the seedlings grown in the reference growth medium, was visible when the biochar concentrations increased.On the other hand, an increasing trend in root length was visible with rising biochar concentrations when seedlings were treated with BC-100-0, with a significantly longer root when 0.25 or 0.50 wt% was added to the reference growth medium.The latter could be explained by the P-deficient environment created by the amendment of BC-100-0 (Fig. S5).Phosphorus is a known macronutrient that plays an important role in plant growth and development and is involved in multiple cellular processes like photosynthesis and energy production 46 .Moreover, a previous study indicated that A. thaliana and other plants change their root architecture depending on the P concentration as  part of their P acquisition strategy 47 .Furthermore, the importance of the P concentration for plant growth in our experiment is indicated by a trend towards significance for the correlation between the P concentration in the medium and the resulting fresh weight (r = 0.43; p = 0.068).In addition, biochar treatment with BC-100-0, BC-75-25 and BC-50-50 significantly increased EI growth (Fig. 4c) and decreased EI defence (Fig. 4d) levels in plants compared to the seedlings grown in the reference growth medium.The decrease in EI defence , an indicator of plant stress, in seedlings treated with BC-100-0 indicates that although the biochar contained a Σ16 EPA PAHs concentration that exceeded the EBC-AGRO threshold (Table 4), these PAHs do not cause PAHs-induced stress in A. thaliana seedlings in this set-up and further confirms that P deficiency in the medium with BC-100-0 is the main reason for the decreased growth.Based on the Σ16 EPA PAHs concentration of current biochars (Table 4), the maximal concentration of Σ16 EPA PAHs in the medium would be only 0.04 mg Σ16 EPA PAHs/L at the highest biochar concentration, in case all the PAHs leach out to the reference growth medium.This concentration is much lower than concentrations used to study PAH toxicity in A. thaliana.For example, multiple studies used phenanthrene to study the effect of PAHs on A. thaliana growth with concentrations ranging between of 17-222 mg/L [48][49][50][51] .Moreover, earlier research already evidenced that only a fraction of the Σ16 EPA PAHs (max.16%) is released from (sewage sludge) biochar (Σ16 EPA PAHs: 1-14 mg/kg), with almost no release of 3 + rings PAHs 52 .Thus, much lower Σ16 EPA PAHs are expected compared to the maximal theoretical concentration (0.04 mg Σ16 EPA PAHs/L).A previous study evidenced acute PHEN-induced stress in A. thaliana at much higher concentrations (approx.17 mg PHEN/L) 48 .This confirms that in the current set-up no PAHs-induced stress in A. thaliana is expected.However, in line with PTEs released from biochar, the persistence and toxicity of PAHs released from biochar should be further assessed under different weathering conditions and at different trophic levels.Furthermore, for the biochars with more CM contribution, i.e.BC-25-75 and BC-0-100, a significant increase of the EI defence concomitantly with no effect or a decrease in EI growth were found in seedlings when treated with 0.50 wt%.This increased stress could be caused by the higher concentrations of K + and Na + found in the medium after biochar BC-25-75 or BC-0-100 incubation (Figure S3).This theory is confirmed by the significant correlations between K + (r = 0.50)/ Na + (r = 0.48) concentration in the medium and the relative seedling's EI defence values.Therefore, the positive effects of treatment with BC-75-25 and BC-50-50 observed regarding fresh weight, www.nature.com/scientificreports/reduced plants stress (EI defence ) and increased plant growth (EI growth ) are believed to come from the right balance between the P and Na + /K + concentration, as these correlate to the fresh weight and EI defence , respectively.The treatment with biochars BC-50-50 and BC-75-25 resulted in the highest P concentrations in the medium, which could also explain the increased plant growth.This indicates that co-pyrolysis can be a technique to promote plant growth through biochar engineering.

Conclusions
In this study, the benefits of co-pyrolysis of CM and TB on the plant growth of A. thaliana were investigated.Our results showed that the biochar composition and toxicity could be controlled through co-pyrolysis.In case of biochar toxicity, up to 50 wt% of CM can be added to the TB without exceeding the EBC-AGRO PTE threshold in the resulting biochar.In the case of the PAHs concentration, no trend was observed, but no biochar, except BC-100-0 and BC-25-75, exceeded the EBC-AGRO threshold.All co-pyrolysis biochars increased the fresh weight and decreased plant stress independent of the used concentrations, except biochars originating from feedstock blends with a high concentration of CM.In contrast, biochars from the original feedstocks showed an increased stress response and/or a reduced plant fresh weight.This could be linked to the leached total P and Na + /K + concentrations as these are correlated to growth and stress parameters.Therefore, the biological outcome can be strongly linked to the biochars' composition.These results suggest that co-pyrolysis can be used to promote plant growth through biochar engineering by modifying the physicochemical properties that decrease the biochar toxicity, while maintaining its positive characteristics, hence improving plant growth.To confirm these results, further research should be carried out to investigate the long-term effects of co-pyrolysis biochar on plant growth and stress response.

Table 2 .
The co-pyrolysis biochars' elemental composition (TC-HNO), the TC/N, O/C and H/C ratios, ash content, macronutrients (P, K, Ca, Mg), pH and electrical conductivity.The values represent the average (standard deviation).